Name: high throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization
Uploaded: 2014-11-27T15:00:00
Description: Watch this Scientific Journal Video about High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization at JoVE.com

The authors would like to thank the following funding sources: RO1 DA 12104, RO1 DA 022935, RO1 DA031202, K05DA033881, P50 DA 011806, 1R01DA034582 (to S.R) and F31 DA026264-01A1, T32 DA07097 (to J.N.).

NOTA: Questo protocollo può essere utilizzato per più applicazioni come la quantificazione della fagocitosi e polimerizzazione usati nel nostro lavoro precedentemente pubblicato 12. Il protocollo si presta ad una varietà di modifica e tipi e particelle Tabella 1 elenca cellulari impiegato con successo in studi precedenti. Uso standard di questo protocollo per la quantificazione di fagocitosi o polimerizzazione è illustrato in Figura 1. <p class="jove_content" fo:keep-...

<ol>
	<li>Murphy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Janeway's Immunobiology. , (2012).</li><li>Burke, D. W., O'Connor, D. O., Zalenski, E. B., Jasty, M., Harris, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromotion+of+cemented+and+uncemented+femoral+components.">Micromotion of cemented and uncemented femoral components.</a> The Journal Of Bone And Joint Surgery. British Volume. 73 (1), 33-37 (1991).</li><li>Duan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+malaria+by+enhanced+phagocytosis+of+erythrocytes+in+LMP7-deficient+mice.">Resistance to malaria by enhanced phagocytosis of erythrocytes in LMP7-deficient mice.</a> PloS One. 8 (3), 59633 (2013).</li><li>Dementhon, K., El-Kirat-Chatel, S., Noel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+in+vitro+model+for+the+multi-parametric+quantification+of+the+cellular+interactions+between+Candida+yeasts+and+phagocytes.">Development of an in vitro model for the multi-parametric quantification of the cellular interactions between Candida yeasts and phagocytes.</a> PloS One. 7 (3), 32621 (2012).</li><li>Al-Bader, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+trehalose+biosynthesis+in+Aspergillus+fumigatus+development,+stress+response,+and+virulence.">Role of trehalose biosynthesis in Aspergillus fumigatus development, stress response, and virulence.</a> Infection And Immunity. 78 (7), 3007-3018 (2010).</li><li>Acosta-Iborra, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+oxygen+sensing+modulates+antigen+presentation+and+phagocytic+functions+involving+IFN-gamma+production+through+the+HIF-1+alpha+transcription+factor.">Macrophage oxygen sensing modulates antigen presentation and phagocytic functions involving IFN-gamma production through the HIF-1 alpha transcription factor.</a> Journal Of Immunology. 182 (5), 3155-3164 (2009).</li><li>Ojielo, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+phagocytosis+and+clearance+of+Pseudomonas+aeruginosa+in+the+lung+following+bone+marrow+transplantation.">Defective phagocytosis and clearance of Pseudomonas aeruginosa in the lung following bone marrow transplantation.</a> Journal Of Immunology. 171 (8), 4416-4424 (2003).</li><li>Neu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD14-dependent+monocyte+isolation+enhances+phagocytosis+of+listeria+monocytogenes+by+proinflammatory,+GM-CSF-derived+macrophages.">CD14-dependent monocyte isolation enhances phagocytosis of listeria monocytogenes by proinflammatory, GM-CSF-derived macrophages.</a> PloS One. 8 (6), 66898 (2013).</li><li>Sokolovska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+caspase-1+by+the+NLRP3+inflammasome+regulates+the+NADPH+oxidase+NOX2+to+control+phagosome+function.">Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function.</a> Nature Immunology. 14 (6), 543-553 (2013).</li><li>Janko, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRP/anti-CRP+antibodies+assembly+on+the+surfaces+of+cell+remnants+switches+their+phagocytic+clearance+toward+inflammation.">CRP/anti-CRP antibodies assembly on the surfaces of cell remnants switches their phagocytic clearance toward inflammation.</a> Frontiers in Immunology. 2 (2), 70 (2011).</li><li>Clatworthy, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+lupus+erythematosus-associated+defects+in+the+inhibitory+receptor+FcgammaRIIb+reduce+susceptibility+to+malaria.">Systemic lupus erythematosus-associated defects in the inhibitory receptor FcgammaRIIb reduce susceptibility to malaria.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (17), 7169-7174 (2007).</li><li>Ninkovic, J., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+decreases+bacterial+phagocytosis+by+inhibiting+actin+polymerization+through+cAMP-,+Rac-1-,+and+p38+MAPK-dependent+mechanisms.">Morphine decreases bacterial phagocytosis by inhibiting actin polymerization through cAMP-, Rac-1-, and p38 MAPK-dependent mechanisms.</a> The American Journal Of Pathology. 180 (3), 1068-1079 (2012).</li><li>Nix, R. N., Altschuler, S. E., Henson, P. M., Detweiler, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemophagocytic+macrophages+harbor+Salmonella+enterica+during+persistent+infection.">Hemophagocytic macrophages harbor Salmonella enterica during persistent infection.</a> PLoS Pathogens. 3 (12), 193 (2007).</li><li>Xue, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+gene+transfer+and+expression+in+cord+blood-derived+CD34++hematopoietic+stem+and+progenitor+cells+by+a+hyperactive+Sleeping+Beauty+transposon+system.">Stable gene transfer and expression in cord blood-derived CD34+ hematopoietic stem and progenitor cells by a hyperactive Sleeping Beauty transposon system.</a> Blood. 114 (7), 1319-1330 (2009).</li><li>Hed, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+distinguishing+ingested+from+adhering+particles.+Methods+in+Enzymology.">Methods for distinguishing ingested from adhering particles. Methods in Enzymology.</a> 132. , 198-204 (1986).</li><li>Scott, A. J., Woods, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+internalization+of+Histoplasma+capsulatum+by+mammalian+cell+lines+using+a+fluorometric+microplate+assay.">Monitoring internalization of Histoplasma capsulatum by mammalian cell lines using a fluorometric microplate assay.</a> Medical Mycology. 38 (1), 15-22 (2000).</li></ol>

I principali limiti della tecnica fluorimetrica sono varianza sperimentale nonché perdita cellulare associata con ampio lavaggio e l&#39;uso di cellule non aderenti. Variance si osserva soprattutto a causa della variazione di particelle fluorescenti come pesatura e pipettaggio durante la preparazione della soluzione madre non è un modo esatto di mantenere numeri identici di particelle da esperimento per esperimento. Per affrontare il problema della varianza tra esperimenti, i dati possono ...

Quantificazione del segnale fluorescente è stato ampiamente utilizzato in una moltitudine di metodi scientifici che vanno dalla PCR, citometria a flusso, microscopia confocale e FRET analisi multiplex ELISA. Fluorescenza imaging e quantificazione ha una vasta applicazione e può essere un ottimo strumento per l&#39;analisi quantitativa dei vari processi cellulari. L&#39;uso di marcatori fluorescenti e il loro segnale è stato rivoluzionato negli ultimi dieci anni, e la comparsa di lettori di piastre fluorescenti ha facilitato l&#39;alta quantificazione throughput fluorescenza emessa durante i processi cellulari. Analisi fluorimetrica può servire come un grande strumento di quantificazione di fagocitosi. Fagocitosi è stato studiato dopo la scoperta dei fagociti da Metchnikoff nel 1800 1. Nel corso degli anni, una varietà di metodi sono stati utilizzati per esaminare questo importante processo essenziale per la difesa immunitaria innata contro l&#39;invasione batterica, virale, fungina e patogeni parassiti 2-5. Metodi precedenti di quantificazione utilizzata microscopia e tecniche stereologici al fine di visualizzare le cellule che sono phagocytosing, che sono stati poi quantificati contando di particelle interiorizzate (manualmente o con l&#39;uso di software) 6-8. Alcuni svantaggi di usare solo la microscopia per l&#39;analisi quantitativa sono che il conteggio manuale dei batteri è lavoro intensivo e più inclini a pregiudizi dell&#39;osservatore. Un altro metodo utilizzato nella quantificazione della fagocitosi è la tecnica microbiologica di placcatura di batteri lisati cellulari (seguenti fagocitosi) su piastre di coltura batterica, ma questo metodo può non tenere conto di meccanismi e presenza di batteri in parte internalizzati battericida. Questo metodo è ancora più alta intensità di lavoro rispetto alla microscopia e richiede diversi giorni per analizzare. Flusso sembra citometria essere il più veloce, più efficace per quantificare fagocitosi ed è stato utilizzato da molti gruppi 9-11, ma il costo elevato comunemente associato con l&#39;instrumento necessario per l&#39;analisi, è il metodo più costoso rispetto ai saggi precedentemente menzionati. Il metodo fluorimetrica è una buona alternativa per citometria a flusso per l&#39;analisi di internalizzazione delle particelle in quanto offre la quantificazione imparziale di apparecchiature di fluorescenza utilizzando tale non è il costo proibitivo. Altri benefici aggiunti di fluorometria sono ad alta efficienza, ed elevate capacità di throughput per quantificare la fluorescenza emessa dalle particelle interiorizzati etichettati. Vantaggi di fluorometria possono essere estrapolati per la quantificazione di processi diversi fagocitosi. Ad esempio, l&#39;analisi fluorimetrica può essere applicato per studiare qualsiasi processo che porta a cambiamenti nell&#39;espressione di intracellulare o recettori di membrana, cambiamenti nella permeabilità delle cellule / redditività, l&#39;efficacia di trasfezione, e la modulazione di polimerizzazione. Uno svantaggio della tecnica fluorimetrica è che, a seconda delle etichette utilizzate, ci può essere un altoesperimento di sperimentare la variabilità che di solito può essere risolto dimostrando dati attraverso la quantificazione relativa, come il cambiamento volte o cento di aumento dal controllo.

<table border="0" cellpadding="0" cellspacing="0" width="820">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:131px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:255px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">1-&#956;m yellow-green fluorescent Fluo-Spheres;</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">F8852</td>
			<td style="width:255px;">combine 50&#956;l with 50&#956;l opsonizing reagent for 30 min at 37oC before use</td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;">Heat killed E. coli BioParticles fluorescein conjugate</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">E2861</td>
			<td style="width:255px;">reconstitute (5 mg) in 50&#956;l of PBS and combine with 50&#956;l opsonizing reagent for 30 min at 37oC before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Opsonizing reagent</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">E2870</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Rhodamine phalloidin</td>
			<td style="width:131px;">Molecular Probes</td>
			<td style="width:119px;">R415</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Trypan blue</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:119px;">15250-061</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">the items below are available in many brands but the items we used in this study are from the following manufacturers</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:66px;width:315px;">RPMI Cell growth media&#160;</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:119px;"></td>
			<td style="width:255px;">Supplemented with 10% FBS and 1% pennicillin/Streptomycin; warm in 37oC before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Fluorescent plate reader-Fluostar Omega</td>
			<td style="width:131px;">BMG Labtech</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Paraformaldehyde (16%)</td>
			<td style="width:131px;">Fischer Scientific</td>
			<td>AA433689M</td>
			<td style="width:255px;">dilute to 4% before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">96 well plates</td>
			<td style="width:131px;">Greiner</td>
			<td style="width:119px;">655097</td>
			<td style="width:255px;">clear or black or clear bottom - black plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Multichannel pipette (8-12 channels)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Reagent reservoirs</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1x PBS</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Microfuge tubes (0.6 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">&#160;Conicles (10 ml)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

high throughput fluorometric technique for assessment of macrophage phagocytosis and actin polymerization

L&#39;obiettivo dell&#39;analisi fluorimetrica è di servire come un efficiente, economico, metodo ad alta velocità di analisi di fagocitosi e di altri processi cellulari. Questa tecnica può essere utilizzata in una varietà di tipi di cellule, sia aderenti e non aderenti, per esaminare una serie di proprietà cellulari. Quando si studia la fagocitosi, tecnica fluorimetrica utilizza tipi di cellule fagocitiche come i macrofagi, e le particelle opsonizzati fluorescente cui fluorescenza può essere spento in presenza di blu trypan. Dopo la placcatura dei macrofagi aderenti in piastre a 96 pozzetti, particelle fluorescenti (verde o rosso) sono amministrati e le cellule sono autorizzati a fagocitare per vari periodi di tempo. Dopo internalizzazione delle particelle fluorescenti, le cellule vengono lavate con trypan blu, che facilita estinzione di segnale fluorescente dai batteri che non sono internalizzati, o sono semplicemente aderendo alla superficie cellulare. Al termine del lavaggio trypan, le cellule sono lavate con PBS, fisso, unnd colorati con DAPI (etichetta nucleare fluorescente blu), che serve a etichettare nuclei delle cellule. Con una semplice quantificazione fluorimetrico attraverso la lettura piatto di nucleare (blu) o particella (rosso / verde) di fluorescenza si può esaminare il rapporto tra unità di fluorescenza relativi di verde: blu e determinare un indice di fagocitosi indicativa della quantità di batteri fluorescenti interiorizzato per cella. La durata del test utilizzando un metodo e multicanale 96 pozzetti pipette per il lavaggio, da fine fagocitosi a fine acquisizione dati, è inferiore a 45 min. Citometria a flusso potrebbe essere utilizzato in modo simile, ma il vantaggio di fluorometria è la sua alta produttività, rapida metodo di valutazione con minima manipolazione dei campioni e veloce quantificazione di intensità fluorescente per cella. Strategie simili possono essere applicati a cellule non aderenti, batteri vivi etichettati, actina polimerizzazione, e sostanzialmente qualsiasi processo che utilizza fluorescenza. Pertanto, fluorimetria è un metodo promettente per il suo basso costo, alta throughpcapacità ut nello studio dei processi cellulari.

Due modi principali di quantificare fagocitosi e la successiva polimerizzazione actina attraverso l&#39;utilizzo di questo protocollo è di osservare la fagocitosi continua o sincronizzato. Analisi microscopica (Figura 1B) mostra immagini fluorescenti paragonabili a ciò che il fluorimetro sta registrando (vedi anche Figura 1). Nella Figura 1B sono fluorescenza rossa di actina, verde 268 fluorescenza FITC etichette HKOP E. coli, mac...

Watch this Scientific Journal Video about High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization at JoVE.com

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Alta Tecnica Throughput fluorometrica per la valutazione dei macrofagi fagocitosi e polimerizzazione

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

high-throughput-fluorometric-technique-for-assessment-macrophage

Research

JoVE Journal

Immunology and Infection

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

High-throughput Metodo di rilevazione per il virus dell&#39;influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Ricerca

Immunologia e infezioni

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

High-throughput screening per largo spettro chimico inibitori di RNA virus

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Macrofagi Colesterolo Depletion e il suo effetto sulla fagocitosi di<em&gt; Cryptococcus neoformans</em

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Un test compatibile high-throughput per valutare Drug efficacia contro macrofagi diversi passaggi<em&gt; Mycobacterium tuberculosis</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

Analisi enzimatica fluorometrica cell-free biochimico per la misura di High-throughput di perossidazione lipidica in lipoproteina ad alta densità

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Valutazione degli effetti di farmaci a base di anticorpi sul midollo osseo murino e l'attivazione dei macrofagi peritoneali

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

Utilizzando Enhanced fluorescenza verde che esprimono proteine Escherichia Coli per valutare la fagocitosi dei macrofagi peritoneali del Mouse

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

Alta velocità effettiva In Vitro valutazione della latenza inversione agenti sulla trascrizione di HIV e Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Fagocitosi del macrofago alveolare e la Clearance di batteri nei topi

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

In Vivo Assessment of Alveolar Macrophage Efferocytosis Following Ozone Exposure

Ozone (O3) is a criteria air pollutant that exacerbates and increases the incidence of chronic pulmonary diseases. O3 exposure is known to induce pulmonary inflammation, but little is known regarding how exposure alters processes important to the resolution of inflammation. Efferocytosis is a resolution process, whereby macrophages phagocytize apoptotic cells. The purpose of this protocol is to measure alveolar macrophage efferocytosis following O3-induced lung injury and inflammation. Several methods have been described for measuring efferocytosis; however, most require ex vivo manipulations. Described in detail here is a protocol to measure in vivo alveolar macrophage efferocytosis 24 h after O3 exposure, which avoids ex vivo manipulation of macrophages and serves as a simple technique that can be used to accurately represent perturbations in this resolution process. The protocol is a technically non-intensive and relatively inexpensive method that involves whole-body O3 inhalation followed by oropharyngeal aspiration of apoptotic cells (i.e., Jurkat T cells) while under general anesthesia. Alveolar macrophage efferocytosis is then measured by light microscopy evaluation of macrophages collected from bronchoalveolar (BAL) lavage. Efferocytosis is finally measured by calculating an efferocytic index. Collectively, the outlined methods quantify efferocytic activity in the lung in vivo while also serving to analyze the negative health effects of O3 or other inhaled insults.

This manuscript describes a protocol for determining whether exposure to ozone, a criteria air pollutant, impairs alveolar macrophage efferocytosis in vivo. This protocol utilizes commonly used reagents and techniques and can be adapted to multiple models of pulmonary injury to determine effects on alveolar macrophage efferocytosis.